2. University of Gondar
Institute of Biotechnology
Techniques in Biotechnology (Biot.602)
Lecture 4
DNA quantification & purity determination
3. 3
Reliable measurement of DNA concentration is important
for many applications
DNA quantity and quality can be assessed using several
different methods include:
Absorbance by spectrophotometer or Nanophotometer.
Agarose gel electrophoresis .
Absorbance: is the most common easies to
determine DNA yield and purity.
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4. Quality of DNA using spectrophotometer
• An instrument employed to measure the amount of
light that a sample absorbs.
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5. 5
The rings of the bases (A, C, G, T, U)
are made up of alternating single
and double bonds.
Such ring structures absorb in
the U.V.
Each of the four nucleotide
bases has a slightly different
absorption spectrum, and
The spectrum of DNA is
the average of them.
Tadele T, April 2010 E.C
6. ◦ DNA UV absorbance at 260nm.
◦ protein >> at 280nm.
◦ Carbohydrate >> at 230nm.
◦ Any insoluble light-scattering components……. absorbance at
320 nm.
Note: Nucleic acids absorb light at 260 nm ,the A260 reading should
be between 0.1–1.0. The spectrophotometer is most accurate when
measurements are in the range of 0.1–1.0.
However, DNA is not the only molecule that can absorb UV-
light at 260nm.
Since RNA also has a great absorbance at 260nm will
contribute to the total measurement at 260nm
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7. The ratio of the absorbance at 260 nm/280 nm is a
measure of the purity of a DNA; it should be between
1.7 and 2.0.
If < 1.7, the nucleic acid preparation may be contaminated with
protein. Use protinase K to remove protein.
If > 2.0 indicates RNA contamination. RNase should be used to
remove the contaminating RNA.
DNA Purity (A260/A280) = (A260 reading – A320 reading)
/(A280 reading – A320 reading)
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8. The ratio of the absorbance at 260 nm/320 nm is a measure
of the purity of a DNA sample from organics and/or salts;
it should be about 2.0.
Low A260/A320 ratio indicates contamination by organics
and/or salts.
The absorbance reading indicates how much the sample is pure.
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9. Quantification of DNA by spectrophotometry.
Using TE buffer as the diluent,
Make an appropriate dilution of your DNA depending on
the size of the cuvettes available (e.g. for 1ml cuvettes,
dilute 10 microliter DNA solution in 990 micro liters of
TE).
Determine the absorbance of DNA at 260 nm using TE as the
reference solution (i.e. as a blank).
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10. Using a conversion factor :
◦ one OD at 260 nm is equivalent to
Multiply the absorbance reading by
the conversion factor and
the dilution factor to find the concentration of nucleic
acid.
Pure DNA Concentration (microg/ml) =
(A260 reading – A320 reading) x dilution factor x 50microg/ml
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11. Total yield is obtained by multiplying the
DNA concentration by the final total purified
sample volume.
DNA Yield (microgram/ml) = DNA
Concentration x Total Sample Volume
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12. Problem. From a small culture, you have purified the DNA of a
recombinant plasmid. Then you have resuspended the DNA in a
volume of 50 µL TE. You dilute 20 µL of the purified DNA sample
into a total volume of 1000 µL distilled water. You measure the
absorbance of this diluted sample at 260 nm and 280 nm and obtain
the following readings.
A260 --- 0 . 5 5 0
A280 - 0 . 3 2 4
a) What is the DNA concentration of the 50 µL plasmid prep?
b) How much total DNA was purified by the plasmid prep
procedure?
c) What is the A260/280 ratio of the purified DNA?
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13. 13
Don’t need dilution
The volume required for measurement 3-5
microliters
The concentration given in nanogram
microliters.
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14. 14
Quality of DNA extracted is assessed using
the following simple protocol:
Mix 3µL of DNA with 12µL of loading
Dye
Load this mixture into a 1% agarose gel
Stain with ethidium bromide
Electrophorese at 70–80 volts, 45–90
minutes.
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15. Checking for Degradation DNA
Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation.
Smearing indicates
DNA degradation or
Too much DNA loaded.
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Good quality DNA should
migrate as a high molecular
weight band, with little or no
evidence of smearing.